The unifying hypothesis of this renewal application is that microvascular endothelial dysfunction is present in type 2 diabetes and in insulin-resistant pre-diabetic states and is characterized by impaired insulin and substrate delivery to capillaries (impaired capillary recruitment) and by decreased nutrients/hormone exchange across the capillary endothelium. During the current award, we demonstrated that insulin-induced microvascular recruitment in skeletal muscle occurs more rapidly and at lower insulin concentrations than increases in total blood flow. We also demonstrated that capillary recruitment within skeletal muscle is impaired in states of induced insulin resistance (FFA or TNF-a infusion, or obesity). This impairment in capillary recruitment was accompanied by diminished insulin-mediated glucose uptake. In Aim 1, we will test whether insulin at physiologically relevant concentrations acts directly on endothelial cells to activate signaling pathways that: a) increase nitric oxide production;b) selectively dilate precapillary arterioles;c) facilitate the trans-endothelial movement of glucose and insulin. In the Aim 2, we will test the hypothesis that insulin resistance, either genetic or provoked by dietary manipulation, is accompanied by microvascular insulin resistance and evidence of microvascular inflammation and that resistance to insulin-mediated capillary recruitment is accompanied by reduced transport of insulin into muscle interstitium. In studies proposed under Aim 3, we will test the hypotheses that: insulin at physiologic relevant concentrations acts both peripherally and centrally (within the CMS) to recruit microvasculature in skeletal muscle. To quantify microvascular recruitment, studies will utilize methods (developed jointly in our laboratories in Virginia and Hobart, Tasmania) for measurement of microvascular volume (contrast-enhanced ultrasound) and capillary surface exchange area (1-MX extraction). To quantify trans-endothelial insulin transport, we will measure both in vivo and in vitro the trans-endothelial flux of fluorescent or radio-labeled insulin. We will define the activity of insulin-signaling and inflammatory pathways using a combination of confocal microscopy and Western blotting methods. These studies, when successfully completed, should provide a more comprehensive understanding of the role of the microvasculature as a site of regulation of insulin action in health and insulin resistant states.